Saudi Journal of Biological Sciences xxx (xxxx) xxx

Contents lists available at ScienceDirect

Saudi Journal of Biological Sciences

journal homepage: www.sciencedirect .com

Original article

HPLC-PDA-MS/MS profiling of secondary metabolites from Opuntiaficus-indica cladode, peel and fruit pulp extracts and their antioxidant,neuroprotective effect in rats with aluminum chloride inducedneurotoxicity

https://doi.org/10.1016/j.sjbs.2020.07.0031319-562X/� 2020 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

⇑ Corresponding author at: AgroBiosciences Research Division, Mohammed VIPolytechnic University, Lot 660–Hay Moulay Rachid, 43150 Ben-Guerir, Morocco.

E-mail address: mansour.sobeh@um6p.ma (M. Sobeh).

Peer review under responsibility of King Saud University.

Production and hosting by Elsevier

Please cite this article as: S. S. El-Hawary, M. Sobeh, W. K. Badr et al., HPLC-PDA-MS/MS profiling of secondary metabolites from Opuntia ficus-inddode, peel and fruit pulp extracts and their antioxidant, neuroprotective effect in rats with aluminum chloride induced neurotoxicityOpuntia ficus->, Saudi Journal of Biological Sciences, https://doi.org/10.1016/j.sjbs.2020.07.003

Seham S. El-Hawary a, Mansour Sobeh b,f,⇑, Wafaa K. Badr c, Mohamed A.O. Abdelfattah d, Zeinab Y. Ali e,Mona E. El-Tantawy c, Mohamed A. Rabeh a, Michael Wink b

aDepartment of Pharmacogosy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egyptb Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120-Heidelberg, GermanycDepartment of Medicinal plants and natural products, National Organization of Drug Control and Research, Giza, EgyptdCollege of Engineering and Technology, American University of the Middle East, KuwaiteDepartment of Biochemistry, National Organization of Drug Control and Research, 12553 Giza, EgyptfAgroBiosciences Research Division, Mohammed VI Polytechnic University, Lot 660–Hay Moulay Rachid, 43150 Ben-Guerir, Morocco

a r t i c l e i n f o

Article history:Received 8 December 2019Revised 30 June 2020Accepted 2 July 2020Available online xxxx

Keywords:Opuntia ficus-indicaHPLC-MS/MSNeurotoxicityAlCl3In vivoMolecular docking

a b s t r a c t

Opuntia ficus-indica (L.) Mill. (OFI), also known as Indian fig Opuntia or prickly pear, is a member of thefamily Cactaceae that produces edible, nutritionally rich sweet fruits. It has been traditionally used totreat several health disorders and is considered to possess various therapeutic properties. In this work,we have characterized 37 secondary metabolites using HPLC-MS/MS, identified the polysaccharide fromthe fruits and cladodes pulp, and estimated the neuroprotective activity. All tested extracts exhibited sub-stantial antioxidant activities in-vitro and neuroprotective potential in AlCl3 induced Alzheimer’s condi-tion. Administration of OFI extracts attenuated AlCl3 induced learning and memory impairment asconfirmed from passive avoidance test and counteracted the oxidative stress as manifested from decrea-sein the elevated MDA level, increased TAC, GSH, SOD and CAT levels. OFI extracts significantly decreasedthe elevated brain levels of proinflammatory cytokines (NF-jb and TNF-a), increased anti-inflammatorycytokine (IL-10), and monoamine neurotransmitters (NE, DA, 5-HT) as compared to positive controlgroup. The extracts showed a significant decrease in acetylcholinesterase level (AChE) as compared withAlCl3. Furthermore, molecular docking was performed to investigate the ability of the major constituentsof OFI extracts to interact with acetylcholinesterase (AChE) and serotonin transporter (SERT). Among thetested extracts, cladodes contain highest phenolic content and exhibited the highest antioxidant, anti-inflammatory and neuroprotective activities, which could be attributed to presence of several polyphe-nols, which could function as AChE and SERT inhibitors. Opuntia ficus-indica might be promising candi-date for treating Alzheimer disease, which makes it a subject for more detailed studies.� 2020 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access

article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Formation and inactivation of reactive oxygen species (ROS) is acommon biological feature in all aerobic organisms. Despite beingrequired for normal cell function and immune response, high con-centrations of ROS lead to oxidative stress, which can damage DNA(induce mutations), proteins and bio membranes. Several diseasesand health conditions in humans are linked to oxidative stressincluding cancer, inflammation, diabetes mellitus, ischemia, pul-monary fibrosis, aging and Alzheimer’s disease (AD). The latter rep-resents a neurodegenerative disorder leading to progressive

ica cla-indica–

2 S.S. El-Hawary et al. / Saudi Journal of Biological Sciences xxx (xxxx) xxx

dementia among the elderly (Feldman and Quenzer, 1984; Dumontand Beal, 2011; Van Wyk and Wink, 2017).

Natural antioxidants that are abundant in several foods andmedicinal plants have a remarkable free radical scavenging poten-tial that could be useful to counteract Alzheimer’s disease andother neurological disorders related to oxidative stress (Chaouchet al., 2016a, Van Wyk and Wink, 2017, 2015; El-Hawary et al.,2018, 2016; El Tanbouly et al., 2017).

Opuntia ficus-indica (L.) Mill., known as Indian fig Opuntia, bar-bary fig, cactus pear, spineless cactus, and prickly pear, is a mem-ber of the family Cactaceae. This cactus has a Mexican origin but isnow widely distributed in arid and semi-arid regions of the Newand Old World. It produces edible, nutritionally rich sweet fruitsand its spathulate stems (called cladodes) are ingredients of Mex-ican cuisine. Fruits and young stems are traditionally utilized totreat hypertension, asthma, edema, diabetes, burns, indigestion,and other health disorders (Chaouch et al., 2016b; Galati et al.,2003; Barba et al., 2017). They also exhibit antioxidant, anticancer,anti-inflammatory and anti-allergic properties (Barba et al., 2017;Ammar et al., 2015; Benayad et al., 2014). Flowers were reportedto possess antioxidant, antimicrobial, anti-ulcer, anti-inflammatory and wound healing activities, which were attributedto isorhamnetin, quercetin and kaempferol glycosides.

The fruits contain phenolic alkaloids (betalains) such as beta-cyanin and betaxanthin, and glycosylated flavonoids that exhibitseveral pharmacological activities. The red and yellow-coloredbetalains which are water-soluble pigments are used as naturalfood colorant (Stintzing et al., 2005; Ahmed et al., 2005). In termsof antioxidant potential, cactus fruits are twice as potent as pears,apples, tomatoes, bananas, white grapes, and almost equipotent tored grapes and grapefruit (Ahmed et al., 2005; Stintzing and Carle,2006).

In the present study, we have characterized the polyphenolicconstituents of extracts from O. ficus-indica cladodes, fruit peeland fruit pulp and investigated their antioxidant and neuroprotec-tive activities.

2. Materials and methods

2.1. Drugs and chemicals

Aluminum chloride (AlCl3�6H2O) from NODCAR (Giza, Egypt),donepezil reference drug from Eva pharma (Cairo, Egypt) (Donaziltablets), DPPH from Alfa Aesar Co., (Karlsruhe, Germany), L-noradrenaline from MP Biomedicals Co., (Eschwege, Germany),dopamine hydrochloride from Sigma Aldrich Co. (Steinheim, Ger-many), and serotonin hydrochloride from Alfa Aesar Co., (Karl-sruhe, Germany) were used. The used neurotransmitters are ofHPLC grade. Folin-ciocalteu reagent for phenolic content obtainedfrom LOBA Chemie PVT., Ltd, India. All other used materials wereof highest grade, commercially available chemicals and solvents.

2.2. Plant material and extraction

Plant samples (5 kg) of Opuntia ficus-indica (cladode peel andfruits) were collected from Orman Botanical garden, Giza, Egypton July 2016. The plant was authenticated by Eng. Trease LabibYousef (National Gene Bank and Orman Botanical Garden Consul-tant). The samples were extracted with 100% methanol (3 � 2 L)at ambient temperature. The extracts were combined, filteredand reduced under vacuum at 40 �C. After freezing at �70 �C, theextracts were lyophilized yielding fine dried powder.

Please cite this article as: S. S. El-Hawary, M. Sobeh, W. K. Badr et al., HPLC-PDdode, peel and fruit pulp extracts and their antioxidant, neuroprotective effect i>, Saudi Journal of Biological Sciences, https://doi.org/10.1016/j.sjbs.2020.07.003

2.3. Preparation and purification of the polysaccharides

The polysaccharides of both fruits and cladodes were preparedaccording to Laidlaw and Percival (1950). Sliced deseeded fruitsand cladode pulps (100 g) were extracted with hot water(3 � 500 ml at 90–95 �C) for 12 h until complete extraction of thepolysaccharides was reached (tested by giving no cloudiness orprecipitate on addition of four volumes of absolute alcohol toone volume of the extract). Polysaccharides were precipitated byaddition of four volumes of absolute alcohol to one volume of eachextract, centrifuged at 10,000 rpm for 15 min and filtered. Theobtained polysaccharides were purified according to White andRao (1953). They were separately re-dissolved in 50 ml waterand the solutions were re-precipitated with 200 ml ethanol. Theproducts were filtered, washed successively with ethanol and etherand then dried in vacuum desiccators over anhydrous calciumchloride to give (2 g) of fruits pulp mucilage representing 2% ofthe dried weight and (1.87 g) of cladode pulp representing 1.87%of the dried weight.

2.4. Hydrolysis of the polysacchrides

The purified polysaccharides were hydrolyzed according toPazur et al. (1986). Each polysaccharide (100 mg) was separatelyacid hydrolyzed by refluxing with 5 ml of 2 M HCl for 2 h in a boil-ing water bath. After hydrolysis, a slightly dark flocculent precipi-tate was obtained in each case and was filtered off. Excesshydrochloric acid was removed from the filtrate by neutralizationwith sodium carbonate solution. The extracts were filtered andevaporated under reduced pressure.

2.5. Chromatographic investigation of the polysaccharides hydrolysate

HPLC analysis was carried out using Agilent chromatographicsystem (series 1200) coupled to refractive index detector andequipped with quaternary pump, degasser along with auto injec-tor. The chromatographic data was acquired by Agilent software.Samples obtained were analyzed by an Aminex-carbohydrateHPX-87 column (L 250 mm � ID 4.6 mm, particle size (5 mm))under isocratic elution with water, injection volume 20 mL. Theflow rate was set to 0.5 ml/min and the temperature was kept at85 �C and 50 �C for the column and the detector respectively. Thequalitative identification was achieved by comparing relativeretention times of the individual sugars to those of authenticsugars.

2.6. HPLC-PDA-Ms/Ms

The phytochemical constituents in the cladodes, fruit peel andpulp extracts were investigated using a ThermoFinnigan LCQ-Duoion trap mass spectrometer (ThermoElectron Corporation, Wal-tham, Ma, USA) with ESI source (ThermoQuest Corporation, Austin,Tx, USA) (Sobeh et al., 2017a). A Discovery HS F5 column(15 cm � 4.6 mm ID, 5 mm particles, Sigma-Aldrich Co Steinheim,Germany) was employed with a ThermoFinnigan HPLC system.The mobile phase was made of water and acetonitrile (ACN)(Sigma-Aldrich GmbH, Steinheim, Germany) (0.1% formic acideach). ACN was 5% at 0 min, then increased to 30% over 60 min,and to 90% within the last 30 min. The column was then recondi-tioned for 10 min to the initial conditions with (5% ACN). The flowrate was maintained at 1 ml/min with a 1:1 split before the ESIsource. The extracts (25 mL) were injected using autosampler sur-veyor ThermoQuest. Xcalibur software (XcaliburTM 2.0.7, ThermoFischer Scientific, Waltham, Ma, USA) controlled the machine.The MS operated in the negative mode as previously reported(Sobeh et al., 2017b).

A-MS/MS profiling of secondary metabolites from Opuntia ficus-indica cla-n rats with aluminum chloride induced neurotoxicityOpuntia ficus-indica–

S.S. El-Hawary et al. / Saudi Journal of Biological Sciences xxx (xxxx) xxx 3

2.6.1. In vitro antioxidant activity evaluationThe DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging

potential of the extracts was determined as previously describedby (Ghareeb et al., 2018).

2.6.2. Estimation of total phenolic content colorimetricallyTotal polyphenols content (TP) of the different parts were deter-

mined following the procedure described by (Kumazawa et al.,2002) and the procedure reported in the (EuropeanPharmacopoeia 4th edition 2002) using Folin-Ciocalteu colorimet-ric method. The TP content was expressed as gallic acid (mg)equivalent (GAE)/g of the dry plant material using standard cali-bration curve.

2.7. Biological evaluation

2.7.1. Acute oral toxicity testThe acute toxicity test for cladode, peel, fruit pulp and polysac-

charide extracted from Opuntia ficus-indica was conducted follow-ing the guidelines of the Economic Co-operation and Development(OECD) organization no. 423, a step-wise procedure with 3 animalsof single sex per step (OECD, 2001). A single daily dose of extracts5, 50, 300, 2000 mg/kg body weight was administered orally, for14 days. All animals were monitored daily for any sign of toxicityor mortality (OECD, 2001).

2.7.2. Experimental designFifty-six adult male Sprague Dawley rats of 180–200 g weight

were used. Animals were obtained from the animal house ofNational Organization for Drug Control and Research, Giza, Egypt.Rats were housed in stainless steel cages and maintained undercontrolled temperature (25 ± 2 �C), humidity (55 ± 5%) and light(12 h on/12 h off) throughout the experiment. They obtained astandard basal diet with free access to water. Rats were acclima-tized for one week before the start of the experiment. In order toevaluate the health status, body weight measurement, examina-tion of physical appearance, and deviation from normal behavioralparameters were performed on daily basis. Furthermore, water andfood intake were measured daily. No signs of abnormal symptomswere observed during the experimental time. No toxic signs/mor-tality were observed over the experiment time. The study was con-ducted in accordance with the ethical guidelines of Faculty ofPharmacy, Cairo University, and performed according to guidelinesof the US National Institutes of Health on animal care (approvalnumber: MP1505). The ethics committee Institutional Animal Careand Use Committee (IACUC) (Cairo University) specificallyapproved the current study (CU/III/F/29/16).

The animals were divided into 7 groups, 8 rats each. Group1received distilled water and served as control. Group 2 received adaily dose of AlCl3 (70 mg/kg, i.p.) freshly dissolved in distilledwater for six weeks (Ali et al., 2016). Group 3 received donepezil(an inhibitor of acetylcholinesterase) at a daily oral dose by gavageof 10 mg/kg body weight (b.w.) (Shin et al., 2018) with daily doseof AlCl3 (70 mg/kg, ip). Groups 4 to 7 received a daily oral dose ofextracts (cladodes, fruit peel, fruit pulp and polysaccharide)(100 mg/kg b.w.) freshly dissolved in distilled water with dailydose of AlCl3 (70 mg/kg, ip). The passive avoidance test was con-ducted at the end of the experiment to evaluate the learning andmemory functions of the experimental rats. For other biologicalmarkers, blood samples were collected under light anesthesia withether from the retro-orbital venous plexus after 6 weeks. The sam-ples were then centrifuged at 3000 rpm for 10 min to obtain theserum and kept frozen for determination of total antioxidantcapacity (TAC). Rats were sacrificed by decapitation under mildether anesthesia and then their brain tissues were dissected,washed with ice-cold saline and were either subjected immedi-

Please cite this article as: S. S. El-Hawary, M. Sobeh, W. K. Badr et al., HPLC-PDdode, peel and fruit pulp extracts and their antioxidant, neuroprotective effect i>, Saudi Journal of Biological Sciences, https://doi.org/10.1016/j.sjbs.2020.07.003

ately for analysis or kept frozen at – 80 �C. The brain was homog-enized in 20 mM potassium phosphate buffer pH 2.5 to give 20%brain homogenate (W/V) and centrifuged at 3000 rpm for 10 min.For the determination of monoamine neurotransmitters, frozenbrain was homogenized in 75% aqueous methanol. The super-natants were used to estimate the concentrations of catecholamine[norepinephrine (NE), dopamine (DA) and serotonin (5-HT)],acetylcholinesterase (AChE), oxidative stress markers (SOD, CAT,GSH), lipid peroxides expressed as malondialdhyde (MDA) andthe inflammatory mediators (NF-jb, IL-10 and TNF-a).

2.7.3. Assessment of learning and memory functionsLearning and memory functions in rats were determined by the

passive avoidance task as behavioral model. The apparatus com-posed of light and dark chambers with a metal grid floor. Bothchambers are separated by a wall that has a door. The animalswere tested on two consecutive days. In the acquirement trial, eachrat was individually placed in the light chamber. Rapidly afterbeing in the dark chamber, an electric shock (40 V, 0.5 Ma for1 s) was delivered to the animal’s feet through the grid. The animalwas directly taken out from the apparatus and returned back to thecage. After 24 h the rat was placed in the light chamber again andthe time taken to enter the dark chamber was recorded as step-through latency. If the animal did not enter the dark chamberwithin a 3-min test period, the test was terminated and the step-through latency was noted as 180 sec (Prema et al., 2016).

2.7.4. Determination of brain monoamine neurotransmitter levelsBrain homogenate was prepared using electric homogenizer in

which one gram of the rat brain was homogenized using 5 ml of75% aqueous methanol then centrifuged for 5 min at 9000 rpm,then the supernatant was filtered using Millex� syringe filter unit(pore size 0.22 lm), Sigma Aldrich, Germany. Determination ofnorepinephrine (NE), dopamine (DA) and serotonin (5-HT) in brainhomogenates was done using HPLC. A reversed phase column(Chromasil ODS C18, 150 � 4.6 ID, 5 mm) was used. A 20 mM potas-sium phosphate solution, pH 2.5 was used as mobile phase. Theflow rate was 1.5 ml/min and the compounds were detected usinga UV detector at 210 nm. NE, DA, 5-HT concentrations were quan-tified using HPLC and a calibration curve was constructed usingexternal standard as described in (Ahmed et al., 2016).

2.7.5. Estimation of acetylcholinesterase (AChE) activityIn the brain tissue homogenate, AChE content was measured

according to the manufacturer’s instructions by using Kits fromBiodiagnostic company, Giza, Egypt. The assay is based on mea-surement of the change in absorbance at 405 nm following theincrease of yellow color produced from thiocholine reacting withdithiobisnitrobenzoate ion (DTNB reagent).

2.7.6. Estimation of oxidative stress markersMarkers of oxidative stress in both serum and brain homoge-

nates were determined by following the instructions of manufac-ture of kits (Biodiagnostic Company, Giza, Egypt). The totalantioxidant capacity (TAC) was quantified in the serum, SOD, lipidperoxidation, catalase (CAT) GSH content were quantified in thebrain homogenates colorimetrically using standard calibrationcurves as described in the manufacture instructions.

2.7.7. Estimation of neuro-inflammation markersFor the determination of the inflammation markers in brain

homogenates, Enzyme-linked Immune Sorbent Assay (ELISA) tech-nique was used to determine the levels of tumor necrosis factoralpha (TNF-a) and Interleukin-10 (IL-10), using Quantikine kit(R&D Systems, USA). Nuclear factor-kappa B (NF-jB) was deter-

A-MS/MS profiling of secondary metabolites from Opuntia ficus-indica cla-n rats with aluminum chloride induced neurotoxicityOpuntia ficus-indica–

4 S.S. El-Hawary et al. / Saudi Journal of Biological Sciences xxx (xxxx) xxx

mined using Biosource kit (California, USA) as described in themanufacture instructions.

2.8. Molecular modeling

To gain information about the molecular mechanism by whichthe studied plant extracts work to enhance learning and memoryfunctions, some of the abundant secondary metabolites in theextracts were docked into two proteins that are renowned to berelevant in (AD) prognosis and cognition functions, namely theacetylcholinesterase and the serotonin transporter protein. Thestructures of the selected compounds were drawn using thebuilder tool of the software molecular operating environment(MOE), 2013.08; Chemical Computing Group Inc., Montreal, QC,Canada, H3A 2R7, 2016. The ionized form of the selected com-pounds was adjusted using the wash option, and then had theirenergy minimized using the default force-field mmff94x. Thestructures of the crystallized recombinant human acetyl-cholinesterase, PDB id: 4EY7 and human serotonin transporter,PDB id: 5I6X altogether with the corresponding bound ligandswere downloaded from protein data bank (www.pdb.org). Prepar-ing the proteins for docking was done by assigning the protonationstate to add the missed hydrogen atoms, geometry, and energyminimization. The default docking protocol that applies trianglematcher placement method and London dG scoring function wasadopted for docking the extracts’ compounds to the binding sitesof the aforementioned proteins retaining 20 poses. The dockingposes were refined by force-field energy minimization applyingLondon dG scoring function (Sobeh et al., 2017a).

2.9. Statistical analysis

The results were expressed as mean ± SE and analyzed by one-way ANOVA using SPSS (version 22). Significant differencesbetween the groups were assessed by Duncan’s new multiple-range test. The level of p < 0.05 was considered significant.

3. Results

3.1. Chemical constituents of Opuntia ficus-indica (OFI) extracts

The chemical constituents of extracts from different plant partsof Opuntia ficus-indica were characterized using HPLC-MS/MS(Fig. 1 and Table 1). In total, 37 secondary metabolites were char-acterized based on retention times, molecular weight, fragmenta-tion pattern, and comparisons with reported data from the plantand authentic compounds. The extracts were rich in phenolic acids,flavonoids and fatty acids.

A series of O-methylated flavonoids dominated the extract.They were detected at [M�H]- m/z 785, 769, 755, 623, and 477and a daughter ion at m/z 315 and assigned to isorhamnetinglucosyl-rutinoside, isorhamnetin rhamnosyl-rutinoside, isorham-netin pentosyl-rutinoside, isorhamnetin rutinoside, and isorham-netin glucoside, respectively (Table 1 and Fig. S1), along with theaglycone isorhamnetin (Benayad et al., 2014).

Several phenolic acids were detected, among them sinapic acidhexoside at [M�H]- m/z 385, eucomic acid at [M�H]- m/z 239, andpiscidic acid at [M - H]- m/z 255. Also, a 7-glucosyl-oxy-5-methylflavone glucoside was tentatively identified at [M�H]- m/z 611and two daughter ions at 251 and 431 (Table 1 and Fig. S1). How-ever, no betalains were detected in the extracts and this might beattributed to their lower concentration.

Please cite this article as: S. S. El-Hawary, M. Sobeh, W. K. Badr et al., HPLC-PDdode, peel and fruit pulp extracts and their antioxidant, neuroprotective effect i>, Saudi Journal of Biological Sciences, https://doi.org/10.1016/j.sjbs.2020.07.003

3.2. Polysaccharide constituents of Opuntia ficus-indica extracts

Polysaccharides were extracted from the fruits and cladodespulp. After hydrolysis we detected six sugars by HPLC. Data areshown in Table 2.

3.2.1. In vitro antioxidant evaluationThe methanol extracts of the different plant parts displayed

in vitro radical scavenging activity; however, the activity of thecladodes surpassed the other extracts followed by that of the peelthen the pulp and finally the polysaccharides, (Table 3). Similaractivities were reported from Italian Opuntia ficus-indica cladodesand others (Petruk et al., 2017).

3.2.2. Estimation of phenolic contentThe methanol extract of cladodes contained higher content of

phenolic compound (236.5 mg (GAE)/g dry wt) followed by fruitspeel extract (165.2 mg (GAE)/g dry wt) and finally fruits pulpextract (53.48 mg (GAE)/g dry wt). The higher polyphenol contentin cladodes extract than the fruits peel and fruits pulp extracts mayexplain the higher activity of cladodes compared to that of fruitspeel and pulp in the pharmacological studies (antioxidant, anti-inflammatory activity and neuroprotective activity).

3.3. Acute oral toxicity test

The obtained result showed that the oral administration of cla-dode, peel, fruit and polysaccharide extracts were non toxic andsafe up to 2000 mg/kg body weight for 14 days. Therefore, one-twentieth of this tolerated dose (i.e 100 mg/kg body weight/day)was chosen for comparative evaluation of the possible neuropro-tective activity of cladode, peel, fruit and polysaccharide extracts.

3.4. Effect of the different extracts on oxidative stress marker levelsin vivo

In rats, administration of AlCl3 for six weeks significantlyreduced the serum total antioxidant capacity (TAC), superoxidedismutase (SOD), reduced glutathione (GSH), catalase (CAT) andincreased malondialdhyde (MDA) levels when compared tountreated control group. Rats treated with the extracts demon-strated a significant increase in TAC, SOD, GSH and CAT levelsand decreased the elevated MDA level when compared to the ADrats. The cladodes extract showed the highest activities followedby the peel then the pulp extracts and finally the cladode polysac-charides (Fig. 2).

3.5. Effect of the extracts on learning and memory functions

In the passive avoidance task, an animal ought to learn toescape or avoid the electric shock exposure in the dark chamber.All the nocturnal animals including rats only choose, by nature, adark environment, but the animal must suppress such tendencyby remembering the electric shock. All the tested extractsimproved memory and significantly increased latency time (timetaken by rats to enter to the dark chamber as compared to AlCl3rats). Cladodes extracts increased latency time better than peeland pulp extracts, and finally cladodes polysaccharides (Fig. 3).

3.6. Effect of extracts on brain monoamine neurotransmitter levels

The levels of norepinephrine (NE), dopamine (DA) and serotonin(5-HT) in brain homogenates were significantly reduced in theAlCl3 group when compared to the untreated control group. Treat-ments of the rats with the extracts significantly restored the cate-cholamine levels (Table 4).

A-MS/MS profiling of secondary metabolites from Opuntia ficus-indica cla-n rats with aluminum chloride induced neurotoxicityOpuntia ficus-indica–

Fig. 1. HPLC-MS/MS profiles of Opuntia ficus-indica methanol extracts. (a) Cladode extract, (b) peel extract, (c) fruit pulp extract.

S.S. El-Hawary et al. / Saudi Journal of Biological Sciences xxx (xxxx) xxx 5

3.7. Effect of extracts on brain acetylcholinesterase (AChE) levels

Acetylcholinesterase is the key enzyme hydrolyzing the neuro-transmitter acetylcholine. The level of acetylcholinesterase wassignificantly increased in AlCl3 group when compared to the con-trol group. All the studied extracts showed a significant decreasein AChE levels counteracting the effect of AlCl3 (Table 5).

3.8. Effect of the extracts on neuro-inflammation markers in brain

Administration of AlCl3 for six weeks strongly elevated thebrain TNF-a and nuclear factor kappa B (NF-jb) levels anddecreased the interleukin-10 (IL-10) levels compared to the controlgroup. All extracts decreased the brain TNF-a and NF-jb andincreased the reduced IL-10 levels (Fig. 4).

3.9. Molecular modeling

The scoring function and the interaction types between thedocked compounds and the amino acid residues of the acetyl-cholinesterase (AChE) and serotonin transporter protein (SERT)are shown in Table 6.

In this study, some of the abundant compounds in the studiedextracts, were docked into the crystal structures of acetyl-cholinesterase (AChE) and serotonin transporter protein (SERT),which function to regulate the neurotransmitters acetylcholineand serotonin respectively at the synapses of nervous system.

Progression of Alzheimer and other neurological disorders isaccompanied by a reduction of acetylcholine in the brain leadingto defects in cholinergic neurotransmission. Thus, inhibitors ofAChE like the alkaloid galanthamine (from Galanthus nivalis) andthe synthetic drug donepezil are used for symptomatic treatmentof AD (Mehta et al., 2012; Muñoz-Torrero, 2008). The human AChEcrystal structure in complex with donepezil has been recently

Please cite this article as: S. S. El-Hawary, M. Sobeh, W. K. Badr et al., HPLC-PDdode, peel and fruit pulp extracts and their antioxidant, neuroprotective effect i>, Saudi Journal of Biological Sciences, https://doi.org/10.1016/j.sjbs.2020.07.003

resolved in which donepezil was shown to afford several polarand hydrophobic interactions with amino acid residues at theenzyme binding site. Hydrophobic interactions with the residuesTrp 86, Trp 286, Phe 338, while the polar interactions (H-bond)involve the residues Asp 74, Tyr 124, Phe 295, Tyr 341, and the crit-ical one to Tyr 337 (Cheung et al., 2012).

The selected compounds from the extracts were able to affordthe majority of the previously reported interactions with an appre-ciable binding free energy reflected by a scoring function range of�32.39 to �17.69 kcal/mol (Table 6). In general, the glucosidederivatives exhibit better scoring functions and interactions withthe amino acid residues at AChE binding site owing to their bulkierstructure (Fig. S2 and Fig. 5). Two of the glucosides, namely, sinapicacid glucoside and 7-glucosyl-oxy-5-methylflavone glucoside wereeven able to interact with the critical Tyr 337 residue revealingstrong AChE inhibitory potential (Fig. 5). Isorhamnetin, quercetin,and kaempferol aglycons have shown similar interaction fashionwith the target enzyme and close scoring function values (Table 6).

Catecholamine and serotonin influence a diversity of neurolog-ical functions and processes in nervous system including cognitionand memory functions (Berger et al., 2009). The reuptake of theseneurotransmitters leads to signaling termination and is mediatedby a family of sodium symporter transporters including serotonintransporter (SERT), dopamine transporter (DAT), and nore-pinephrine transporter (NET). Drugs interfering with these trans-porters elevate biogenic amines brain level in the synaptic cleftand thus used to alleviate many psychiatric disorders (Bergeret al., 2009; Bröer and Gether, 2012).

Recently, the human SERT crystallographic X-ray structure incomplex with the antidepressants paroxetine and escitalopramhas been reported and revealed central and allosteric binding sites(Coleman et al., 2016). Paroxetine binds only to the central site andaffords different hydrogen bond and hydrophobic interactions withseveral amino acids, most importantly, Asp 98, Tyr 95, Tyr 176, Ile

A-MS/MS profiling of secondary metabolites from Opuntia ficus-indica cla-n rats with aluminum chloride induced neurotoxicityOpuntia ficus-indica–

Table 1Chemical composition of Opuntia ficus-indica extracts.

Tentatively identified compounds Rt (min) [M�H]- MS/MS Extract Ref.

No. Cladodes Fruit peel Fruit pulp

1 Quinic acida 1.38 191 0.10 5.87 0.842 Malic acida 1.66 133 115 3.02 1.99 0.013 Piscidic acida,b 1.95 255 193, 165 7.02 10.40 9.67 (Petruk et al., 2017)4 Diferuloyl-syringsic acidb 2.17 549 255 23.37 10.40 9.67 (Benayad et al., 2014)5 Eucomic acida,b 2.78 239 149, 179, 221 11.62 10.40 16.25 (Petruk et al., 2017)6 Dicaffeoylferulic acidb 3.02 517 239 5.81 17.48 16.25 (Benayad et al., 2014)7 p-Coumaric acid 3-O-glucosidea 4.06 325 163 0.44 16.34 Nd8 7-Glucosyl-oxy-5-methyl flavone glucoside 14.70 611 251, 431 0.04 0.45 Nd9 Sinapic acid 3-O-glucosidea 14.91 385 223 0.40 Nq 8.74 (Sobeh et al., 2017a)10 Sinapic acid 3-O-galactosidea 15.63 385 223 0.31 Nq Nd11 Quercetin pentosyl-rutinosideb 26.02 741 271, 301, 609 0.32 0.15 0.6412 Kaempferol rhamnosyl-rutinoside 26.16 739 255, 285, 593 0.32 0.8413 Isorhamnetin glucosyl-rutinosideb 26.7 785 300, 315, 623 1.9 0.04 1.79 (Benayad et al., 2014)14 Rhamnetin rhamnosyl-rutinosideb 27.87 769 315, 477, 623 7.86 1.40 Nd (Benayad et al., 2014)15 Isorhamnetin rhamnosyl-rutinosideb 29.08 769 315, 477, 623 7.86 2.1 Nd (Benayad et al., 2014)16 Isorhamnetin pentosyl-rutinosideb 29.20 755 315, 461, 623 5.54 7.37 8.85 (Benayad et al.. 2014)17 Quercetin rutinoside (rutin) a 29.36 609 179, 271, 301 20.24 0.98 Nd18 Kaempferol pentosyl-rutinosideb 29.50 725 255, 285, 593 25.26 Nq Nd (Benayad et al., 2014)19 Isorhamnetin pentosyl-rutinoside 30.00 755 315, 461, 623 3.5 Nq 1020 Isorhamnetin pentosyl-hexosideb 31.91 609 179, 300, 315 2.36 1.97 1.48 (Benayad et al., 2014)21 Isorhamnetin rutinosidea,b 34.68 623 179, 300, 315 16.7 29.7 Nd (Benayad et al., 2014)22 Rhamnetin 3-O-glucosidea,b 35.71 477 151, 179, 315 2.88 0.84 1.67 (Benayad et al., 2014)23 Isorhamnetin 3-O-glucosidea,b 36.78 477 151, 179, 315 2 0.84 Nd (Benayad et al., 2014)24 Isorhamnetin coumaroyl-rutinosideb 47.85 785 271, 314, 623 4.37 0.05 Nd (Benayad et al., 2014)25 Rhamnetina 55.69 315 179, 300 1.6 Nq Nd26 Isorhamnetina 61.15 315 179, 300, 315 9.2 3.3 2.927 Diosmetina 63.96 299 179, 271, 284 5.9 Nq Nd28 Tricina 65.00 329 314, 329 21.3 4.23 Nd29 Unknown 65.68 555 225, 299, 555 13.6 6.57 7.1430 Unknown 78.69 513 253, 277, 513 4.7 Nq 0.0131 Hydroxyl octadecadienoic acid 79.4 295 171, 295 1.81 Nq Nd32 Unknown 80.03 515 253 1.72 Nq Nd33 Eicosanoic acida 81.68 311 311 8.5 12.20 Nd34 Eicosanoic acid isomer 82.95 311 311 9 15.19 Nd35 Heneicosanoic acid 84.05 325 325 12.49 6.94 7.6536 Eicosanoic acid isomer 85.31 311 311 8.5 4.23 Nd37 Behenic acida 86.76 339 339 10.59 6.55 Nd

Nq: not quantified, Nd: not detected.a Compounds were confirmed using authentic compounds.b Compounds were previously described from the plant.

Table 2Polysaccharides hydrolysate of Opuntia ficus-indica mucilage from fruit and cladodespulp.

Identified sugar Percentage in mucilage

Fruit pulp Cladodes pulp

Glucuronic acid 77.79 73.11Galacturonic acid 5.35 5.63Glucose 0.036 0.063Galactose 0.097 0.12Rhamnose 0.19 0.13Fructose 0.33 0.31Unidentified 16.21 20.63

Table 3Antioxidant potential of OFI extracts compared to the positive control vitamin C.

Extract DPPH [IC50, mg/mL]

Cladode extract 6.70 ± 0.034Fruit peel extract 10.65 ± 0.054Fruit pulp extract 16.57 ± 0.078Polysaccharides 74.13 ± 0.092Vitamin C 3.15 ± 0.082

6 S.S. El-Hawary et al. / Saudi Journal of Biological Sciences xxx (xxxx) xxx

172, Phe 341, Phe 335, and Thr 497. Escitalopram affords similarinteractions with the central site and binds also to the allostericsite through interacting with the amino acids Asp 328, Arg 104,Gln 332, Ala 331, and Phe 556 (Coleman et al., 2016).

Please cite this article as: S. S. El-Hawary, M. Sobeh, W. K. Badr et al., HPLC-PDdode, peel and fruit pulp extracts and their antioxidant, neuroprotective effect i>, Saudi Journal of Biological Sciences, https://doi.org/10.1016/j.sjbs.2020.07.003

As shown in Table 6, the selected compounds from the extractshave been able to bind to SERT binding sites with a scoring func-tion range of �17.84 to �12.03 kcal/mol. The free aglycones alongwith isorhamnetin glucoside have bound to the central binding siteaffording similar interactions reported by paroxetine. The sinapicacid glucoside has interacted with amino acid residues at the allos-teric site only, while 7-glucosyl-oxy-5-methylflavone glucosidehas fitted mainly in the allosteric site and extended to afford someinteractions with residues in the central binding site (Fig. S2).

Because these compounds are partly ionized at the physiologi-cal pH we have considered docking the ionized form (phenolateion) to AChE and SERT binding sites which revealed ionic interac-tion with basic amino acids such as arginine and lysine (Fig. S3).

4. Discussion

The phytochemical constituents of different parts from Opuntiaficus-indica were characterized using HPLC-MS/MS and their neu-roprotective activity against AlCl3 induced neurotoxicity in ratswas evaluated. A total of 37 secondary metabolites, mainlyisorhamnetin glycosides, were identified. A daily oral dose of100 mg/kg of the extracts for six weeks was able to counteractthe AlCl3 induced neuro-inflammation and oxidative stress. Theyrestore the brain monoamine neurotransmitter and acetyl-cholinesterase levels and enhance learning and memory functionsof the experimental animals.

A-MS/MS profiling of secondary metabolites from Opuntia ficus-indica cla-n rats with aluminum chloride induced neurotoxicityOpuntia ficus-indica–

Fig. 2. Effect of OFI extracts on oxidative stress markers in AlCl3 rats. (a) Total antioxidant capacity (TAC), (b) reduced glutathione (GSH), (c) Malondialdhyde (MDA), (d)superoxide dismutase (SOD), (e) catalase (CAT). Values are expressed as mean ± SE for 8 rats in each group, ap < 0.05 versus control and bp < 0.05 versus AlCl3 group.

Table 4Effects of OFI extracts on brain neurotransmitter levels in AlCl3 rats: serotonin,dopamine, norepinephrine. Cladodes extract showed approximately similar activitieslike the reference drug donepezil.

Groups Noradrenalin Dopamine Serotonin

(mg/g brain)

Control 0.62 ± 0.06 1.68 ± 0.05 0.54 ± 0.03AlCl3 0.33 ± 0.02a 1.0 ± 0.04a 0.25 ± 0.02a

AlCl3 + Donepezil 0.59 ± 0.03b 1.6 ± 0.1b 0.46 ± 0.03b

AlCl3 + Cladode extract 0.57 ± 0.03b 1.53 ± 0.05b 0.44 ± 0.03b

AlCl3 + Peel extract 0.48 ± 0.03b 1.37 ± 0.06b 0.41 ± 0.03b

AlCl3 + Fruits extract 0.44 ± 0.03b 1.25 ± 0.06b 0.38 ± 0.03b

AlCl3 + Polysaccharides 0.40 ± 0.03b 1.11 ± 0.07b 0.33 ± 0.03b

Values are shown as mean ± SE for 8 rats in each group.a p < 0.05 versus control.b p < 0.05 versus AlCl3 group.

Table 5Effect of OFI extracts on acetylcholinesterase (AChE) levels in AlCl3 rats.

Groups AChE (ng/g brain)

Control 0.82 ± 0.07AlCl3 4.48 ± 0.44a

AlCl3 + Donepezil 0.79 ± 0.05b

AlCl3 + Cladode extract 0.90 ± 0.05b

AlCl3 + Peel extract 1.98 ± 0.16a,b

AlCl3 + Fruits extract 3.05 ± 0.22a,b

AlCl3 + Polysaccharides 4.14 ± 0.32a

Data are shown as mean ± SE for 8 rats in each group.a p < 0.05 versus control.b p < 0.05 versus AlCl3 group.

Fig. 3. Effect of OFI extracts on latency time in AlCl3 rats. Cladode and fruit peelextracts exhibited similar activities like the drug donepezil. Data are shown asmean ± SE for 8 rats in each group. ap < 0.05 versus control and bp < 0.05 versusAlCl3 group.

S.S. El-Hawary et al. / Saudi Journal of Biological Sciences xxx (xxxx) xxx 7

Please cite this article as: S. S. El-Hawary, M. Sobeh, W. K. Badr et al., HPLC-PDdode, peel and fruit pulp extracts and their antioxidant, neuroprotective effect i>, Saudi Journal of Biological Sciences, https://doi.org/10.1016/j.sjbs.2020.07.003

The potential of flavonoids (as glucosides or free aglycones) andpolyphenols to transverse the blood brain barrier has been widelyinvestigated. Compounds like catechin, quercetin, naringenin,chrysanthemin (cyaniding-3-glucoside), and others were reportedto be capable of crossing blood brain barrier in cell models andin vivo experiments (Youdim et al., 2003; Faria et al., 2010). Thepossible mechanisms include passive diffusion and active trans-port - by virtue of the glucose transporters - which is mainlyrestricted to glucosides owing to the glucose moiety in their struc-ture (Faria et al., 2009).

AlCl3 can induce biochemical and neurochemical abnormalitiesincluding oxidative brain injury, induction of apoptosis and neu-ronal damage, neuralgia inflammatory reaction, and reduced neu-rotransmitter biosynthesis (Singla and Dhawan, 2014, Kim et al.,2018). The administered dose of the extracts resulted in significantincrease in latency time compared to AlCl3 rats.

A-MS/MS profiling of secondary metabolites from Opuntia ficus-indica cla-n rats with aluminum chloride induced neurotoxicityOpuntia ficus-indica–

Table 6Docking scoring functions (kcal/mol) and compound-amino acid residues interactions in acetylcholinesterase and serotonin transporter proteins.

Compound Acetylcholinesterase Serotonin Transporter Protein

Scoring function (kcal/mol) Amino acids interactions Scoring function (kcal/mol) Amino acids interactions

Sinapic acid glucoside �20.68 Trp 286: H-bondTyr 337: H-bondTyr 341: H-bondTrp 86: HydrophobicTrp 286: Hydrophobic

� 13.02 Arg 104: H-bondGln 332: H-bondLys 490: H-bondGlu 494: Hydrophobic

Isorhamnetin �18.31 Ser 293: H-bondArg 296: H-bondTyr 341: Hydrophobic

� 12.39 Phe 335: H-bondIle 172: HydrophobicTyr 176: HydrophobicGly442: Hydrophobic

Isorhamnetin glucoside �27.49 Tyr 72: H-bondAsp 74: H-bondPhe 297: H-bondTrp 86: HydrophobicTrp 286: Hydrophobic

� 17.84 Tyr 176:H-bondPhe 335: H-bondThr 497: H-bondTyr 95: HydrophobicTyr 176: Hydrophobic

Quercetin �19.75 Asp 74: H-bondThr 83: H-bondPhe 338: HydrophobicTyr 341: Hydrophobic

� 13.66 Tyr 176: HydrophobicPhe 341: HydrophobicGly442: Hydrophobic

Kaempferol �17.69 Tyr 72: H-bondAsp 74: H-bondTyr 341: H-bondSer 293: H-bondArg 296: H-bond

� 12.03 Ile 172: HydrophobicTyr 176: HydrophobicGly 442: Hydrophobic

7-glucosyl-oxy-5-methylflavone glucoside �32.39 Tyr 341: H-bondSer 293: H-bondPhe 295: H-bondTrp 86: HydrophobicTyr 337: HydrophobicTyr 341: Hydrophobic

� 14.22 Asp 98: H-bondArg 104: H-bondThr 497: H-bondPhe 556: Hydrophobic

Fig. 4. Effects of OFI extracts on brain neuro-inflammation marker levels in AlCl3 rats. (a) Tumor necrosis factor (TNFa), (b) nuclear factor kappa B (NF-jb) level, (c)interleukin-10 (IL-10) level. Values are represented as mean ± SE for 8 rats in each group. ap < 0.05 versus control and bp < 0.05 versus AlCl3 group.

8 S.S. El-Hawary et al. / Saudi Journal of Biological Sciences xxx (xxxx) xxx

Monoamine neurotransmitters, namely NE, DA and 5-HT areconsidered to be closely involved in memory and learning. Defi-ciency in the level of these neurotransmitters in the hippocampusleads to the cerebral impairment and plays a role in early patholog-ical process of AD (Singh et al., 2013). The levels of monoamineneurotransmitters were significantly reduced in AlCl3 rats as com-pared to the untreated control group. Significant restoration in thelevels of NE, DA and 5-HT was observed in rats treated with theextracts revealing an improved memory function. Acetylcholineis a crucial neurotransmitter involved in regulating cognitive func-tion. Therefore, the inhibition of AChE is considered a substantial

Please cite this article as: S. S. El-Hawary, M. Sobeh, W. K. Badr et al., HPLC-PDdode, peel and fruit pulp extracts and their antioxidant, neuroprotective effect i>, Saudi Journal of Biological Sciences, https://doi.org/10.1016/j.sjbs.2020.07.003

strategy for treating neurological disorders. The level of AChEwas significantly elevated in AlCl3 rats as compared to theuntreated control group. Significant reduction in the level of AChEwas observed in rats treated with the extracts revealing promisingneuroprotective activities.

Oxidative stress is a major factor contributing to the initiationand progress of several neurological disorders, among them AD(Dumont and Beal, 2011). All OFI extracts were able to reducethe AlCl3 induced oxidative stress in the brain where they demon-strated a significant decrease in oxidative stress marker MDA leveland significant increase in TAC, GSH, SOD and CAT levels revealing

A-MS/MS profiling of secondary metabolites from Opuntia ficus-indica cla-n rats with aluminum chloride induced neurotoxicityOpuntia ficus-indica–

Fig. 5. Docking of isorhamnetin glucoside (3D: top left, 2D: bottom left) and 7-glucosyl-oxy-5-methylflavone glucoside (3D: top right, 2D: bottom right) to AChE binding site.The docked compounds are labeled green, while the co-crystallized reference drug donepezil is cyan blue.

S.S. El-Hawary et al. / Saudi Journal of Biological Sciences xxx (xxxx) xxx 9

substantial antioxidant activities in vivo. Similar activities werereported from OFI fruits and were attributed to the antioxidantaction of the extract (Ha et al., 2003).

Beside oxidative stress, neuro-inflammation is an early event inAD. All the studied extracts demonstrated significant decrease inbrain NF-jb and TNF-a levels and significant increase in IL-10levels. Our results suggest that OFI decrease the AlCl3 inducedneuro-inflammation in the brain. Similar properties were shownby (Kim et al., 2010; Kim et al., 2006) where OFI extracts attenu-ated neuronal injury in rats of cerebral ischemia.

Molecular modeling study provided an insight about the molec-ular targets, which could be plausibly involved in the mechanismof action of the studied OFI extracts. Docking results suggested thatthe extracts’ components could serve as AChE and SERT inhibitorswhich come in agreement with a previous study that has deter-mined the AChE inhibitory activity of some OFI extracts obtainedby various extraction methods and reported IC50 value of0.78 mg/ml for the ethanol extract (Ressaissi et al., 2016). In thesame context, certain quercetin and isorhamnetin glucosides havealso reported to exhibit appreciable activity (Olennikov et al.,2017). Moreover, several studies have reported the inhibitoryeffect of various polyphenols of different sources like green teaand licorice on the monoamine transporter proteins (Olennikovet al., 2017; Ofir et al., 2003; Yáñez et al., 2006).

5. Conclusions

Utilizing LC-MS/MS, 37 secondary metabolites were tentativelyidentified in Opuntia ficus-indica extracts. In DPPH assays, theextracts exhibited substantial antioxidant activities. In vivo, theextracts demonstrated substantial neuroprotective activity againstAlCl3 induced neurotoxicity. In conclusion, Opuntia ficus-indicacounteracts oxidative stress and might be an interesting candidatefor the treatment of several neurological disorders, which needs tobe studied in more detail in other experimental systems.

Please cite this article as: S. S. El-Hawary, M. Sobeh, W. K. Badr et al., HPLC-PDdode, peel and fruit pulp extracts and their antioxidant, neuroprotective effect i>, Saudi Journal of Biological Sciences, https://doi.org/10.1016/j.sjbs.2020.07.003

Declaration of Competing Interest

The authors declare that they have no known competing finan-cial interests or personal relationships that could have appearedto influence the work reported in this paper.

Acknowledgements

We acknowledge the financial support by UM6P for fundingOpen Access Publishing.

References

Ahmed, F., Omar, A.H., Ahmed, Rania F., Saleh, Dalia O., 2016. Combination ofresveratrol and fluoxetine in an acute model of depression in mice: preventionof oxidative DNA fragmentation and monoamines degradation. J. Appl.Pharmaceut. Sci. 1, 001–007.

Ahmed, M., El Tanbouly, N., Islam, W., Sleem, A., El Senousy, A., 2005.Antiinflammatory flavonoids from Opuntia dillenii (Ker-Gawl) Haw. flowersgrowing in Egypt. Phytother. Res. 19, 807–809.

Ali, A.A., Ahmed, H.I., Zaky, H.S., 2016. Stress as a risk factor in induction andprogression of alzheimer’s disease: Impact on the possible protection usingepigallocatechin-3-gallate and/or diazepam. Alzheimer’s Dement 12, 1027–1028.

Ammar, I., Ennouri, M., Attia, H., 2015. Phenolic content and antioxidant activity ofcactus (Opuntia ficus-indica L.) flowers are modified according to the extractionmethod. Ind. Crops Prod. 64, 97–104.

Barba, F.J., Putnik, P., Bursac Kovacevic, D., Poojary, M.M., Roohinejad, S., Lorenzo, J.M., Koubaa, M., 2017. Impact of conventional and non-conventional processingon prickly pear (Opuntia spp.) and their derived products: From preservation ofbeverages to valorization of by-products. Trends Food Sci. Technol. 67, 260–270.

Benayad, Z., Martinez-Villaluenga, C., Frias, J., Gomez-Cordoves, C., Es-Safi, N.E.,2014. Phenolic composition, antioxidant and anti-inflammatory activities ofextracts from Moroccan Opuntia ficus-indica flowers obtained by differentextraction methods. Ind. Crops Prod. 62, 412–420.

Berger, M., Gray, J.A., Roth, B.L., 2009. The expanded biology of serotonin. Annu. Rev.Med. 60, 355–366.

Bröer, S., Gether, U., 2012. The solute carrier 6 family of transporters. Br. J.Pharmacol. 167, 256–278.

Chaouch, M.A., Hafsa, J., Rihouey, C., Le Cerf, D., Majdoub, H., 2016a. Effect ofextraction conditions on the antioxidant and antiglycation capacity of

A-MS/MS profiling of secondary metabolites from Opuntia ficus-indica cla-n rats with aluminum chloride induced neurotoxicityOpuntia ficus-indica–

10 S.S. El-Hawary et al. / Saudi Journal of Biological Sciences xxx (xxxx) xxx

carbohydrates from Opuntia robusta cladodes. Int. J. Food Sci. Technol. 51, 929–937.

Chaouch, M.A., Hafsa, J., Rihouey, C., Le Cerf, D., Majdoub, H., 2016b. Effect of pHduring extraction on the antioxidant and antiglycated activities ofpolysaccharides from opuntia ficus indica. J. Food Biochem. 40, 316–325.

Cheung, J., Rudolph, M.J., Burshteyn, F., Cassidy, M.S., Gary, E.N., Love, J., Franklin, M.C., Height, J.J., 2012. Structures of human acetylcholinesterase in complex withpharmacologically important ligands. J. Med. Chem. 55, 10282–10286.

Coleman, J.A., Green, E.M., Gouaux, E., 2016. X-ray structures and mechanism of thehuman serotonin transporter. Nature 532, 334–339.

Dumont, M., Beal, M.F., 2011. Neuroprotective strategies involving ROS inAlzheimer disease. Free Radical Biol. Med. 51, 1014–1026.

El Hawary, S.S., Saad, S., El Halawany, A.M., Ali, Z.Y., El Bishbishy, M., 2016. Phenoliccontent and anti-hyperglycemic activity of pecan cultivars from Egypt. Pharm.Biol. 54, 788–798.

El-Hawary, S.S., Ali, Z.Y., Younis, I.Y., 2018. Hepatoprotective potential ofstandardized Ficus species in intrahepatic cholestasis rat model: Involvementof nuclear factor-jB, and farnesoid X receptor signaling pathways. J.Ethnopharmacol. https://doi.org/10.1016/J.JEP.2018.11.026.

El Tanbouly, N., El Sayed, A.M., Ali, Z.Y., Abdel Wahab, S., El Gayed, S.H., Ezzat, S.M.,El Senousy, A.S., Choucry, M.A., Abdel-Sattar, E., 2017. Antidepressant-like effectof selected Egyptian cultivars of flaxseed oil on a rodent model of postpartumdepression. Evidence Based Complement. Alternative Med. 2017. https://doi.org/10.1155/2017/6405789.

European Pharmacopeia, 2002. 4th Ed. Council of Europe Press, Strasburg.Faria, A., Pestana, D., Azevedo, J., Martel, F., de Freitas, V., Azevedo, I., Mateus, N.,

Calhau, C., 2009. Absorption of anthocyanins through intestinal epithelial cells–Putative involvement of GLUT2. Mol. Nutr. Food Res. 53, 1430–1437.

Faria, A., Pestana, D., Teixeira, D., Azevedo, J., Freitas, V., Mateus, N., Calhau, C., 2010.Flavonoid transport across RBE4 cells: a blood-brain barrier model. Cell. Mol.Biol. Lett. 15, 234–241.

Feldman, R.S., Quenzer, L.F., 1984. Fundamentals of Neuropsychopharmacology.Sinauer Associates Sunderland, MA.

Galati, E.M., Mondello, M.R., Giuffrida, D., Dugo, G., Miceli, N., Pergolizzi, S., Taviano,M.F., 2003. Chemical characterization and biological effects of Sicilian Opuntiaficus-indica (L.) Mill. fruit juice: antioxidant and antiulcerogenic activity. J. Agric.Food. Chem. 51, 4903–4908.

Ghareeb, M.A., Mohamed, T., Saad, A.M., Refahy, L.A.-G., Sobeh, M., Wink, M., 2018.HPLC-DAD-ESI-MS/MS analysis of fruits from Firmiana simplex (L.) andevaluation of their antioxidant and antigenotoxic properties. J. Pharm.Pharmacol. 70, 133–142.

Ha, H.J., Kwon, Y.S., Park, S.M., Shin, T., Park, J.H., Kim, H.C., Kwon, M.S., Wie, M.B.,2003. Quercetin attenuates oxygen–glucose deprivation-and excitotoxin-induced neurotoxicity in primary cortical cell cultures. Biol. Pharm. Bull. 26,544–546.

Kim, J.-H., Park, S.-M., Ha, H.-J., Moon, C.-J., Shin, T.-K., Kim, J.-M., Lee, N.-H., Kim, H.-C., Jang, K.-J., Wie, M.-B., 2006. Opuntia ficus-indica attenuates neuronal injury inin vitro and in vivo models of cerebral ischemia. J. Ethnopharmacol. 104, 257–262.

Kim, J.M., Kim, D.H., Park, S.J., Park, D.H., Jung, S.Y., Kim, H.J., Lee, Y.S., Jin, C., Ryu, J.H., 2010. The n-butanolic extract of Opuntia ficus-indica var. saboten enhanceslong-term memory in the passive avoidance task in mice. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 34, 1011–1017.

Kim, H.J., Jung, S.W., Kim, S.Y., Cho, I.H., Kim, H.C., Rhim, H., Kim, M., Nah, S.Y., 2018.Panax ginseng as an adjuvant treatment for Alzheimer’s disease. J. Ginseng Res.,1–11 https://doi.org/10.1016/j.jgr.2017.12.008.

Kumazawa, S., Tanguchi, M., Suzuki, Y., Shimura, M., Kwon, M.S., Nakayama, T.,2002. Antioxidant activity of polyphenols in carob pods‘‘. J. Agric. Food. Chem.50 (2), 373–377.

Laidlaw, R., Percival, E., 1950. 101. Studies of seed mucilage. Part V. Examination of apolysaccharide extracted from the seeds of Planta goovata Forsk by hot water. J.Chem. Soc., 528–534

Mehta, M., Adem, A., Sabbagh, M., 2012. New acetylcholinesterase inhibitors forAlzheimer’s disease. Int. J. Alzheimer’s Dis.

Please cite this article as: S. S. El-Hawary, M. Sobeh, W. K. Badr et al., HPLC-PDdode, peel and fruit pulp extracts and their antioxidant, neuroprotective effect i>, Saudi Journal of Biological Sciences, https://doi.org/10.1016/j.sjbs.2020.07.003

Muñoz-Torrero, D., 2008. Acetylcholinesterase inhibitors as disease-modifyingtherapies for Alzheimer’s disease. Curr. Med. Chem. 15, 2433–2455.

OECD, 2001. OECD/OCDE 423. OECD guideline for testing of chemicals acute oraltoxicity.

Ofir, R., Tamir, S., Khatib, S., Vaya, J., 2003. Inhibition of serotonin re-uptake bylicorice constituents. J. Mol. Neurosci. 20, 135–140.

Olennikov, D.N., Kashchenko, N.I., Chirikova, N.K., Akobirshoeva, A., Zilfikarov, I.N.,Vennos, C., 2017. Isorhamnetin and quercetin derivatives as anti-acetylcholinesterase principles of marigold (Calendula officinalis) flowers andpreparations. Int. J. Mol. Sci. https://doi.org/10.3390/ijms18081685.

Pazur, J., Chaplin, N., Kennedy, J., 1986. Carbohydrate Analysis: A PracticalApproach. Oxford Press, London.

Petruk, G., Di Lorenzo, F., Imbimbo, P., Silipo, A., Bonina, A., Rizza, L., Piccoli, R.,Monti, D.M., Lanzetta, R., 2017. Protective effect of Opuntia ficus-indica L.cladodes against UVA-induced oxidative stress in normal human keratinocytes.Bioorg. Med. Chem. Lett. 27, 5485–5489.

Prema, A., Thenmozhi, A.J., Manivasagam, T., Essa, M.M., Akbar, M.D., Akbar, M.,2016. Fenugreek seed powder nullified aluminium chloride induced memoryloss, biochemical changes, a b burden and apoptosis via regulating Akt / GSK3 bSignaling. PLOS one 1, 1–19.

Ressaissi, A., Attia, N., Falé, P., Pacheco, R., Teixeira, V., Machuqueiro, M., Borges, C.,Serralheiro, M., 2016. Aqueous extracts from nopal (Opuntia ficus-indica): antiacetylcholinesterase and antioxidant activity from phenolic bioactivecompounds. Int. J. Green Herbal Chem. 5, 337–348.

Shin, C.Y., Kim, H.S., Cha, K.H., Won, D.H., Lee, J.Y., Jang, S.W., Sohn, U.D., 2018. Theeffects of Donepezil, an acetylcholinesterase inhibitor, on impaired learning andmemory in rodents. Biomol. Therap. 26, 274.

Singh, C., Bortolato, M., Bali, N., Godar, S.C., Scott, A.L., Chen, K., Thompson, R.F., Shih,J.C., 2013. Cognitive abnormalities and hippocampal alterations in monoamineoxidase A and B knockout mice. Proc. Natl. Acad. Sci. 110, 12816–12821.

Singla, N., Dhawan, D.K., 2014. Influence of zinc on calcium-dependent signaltransduction pathways during aluminium-induced neurodegeneration. Mol.Neurobiol. 50, 613–625. https://doi.org/10.1007/s12035-014-8643-7.

Sobeh, M., Hassan, S., El Raey, M., Khalil, W., Hassan, M., Wink, M., 2017a.Polyphenolics from Albizia harveyi exhibit antioxidant activities and counteractoxidative damage and ultra-structural changes of cryopreserved bull semen.Molecules 22, 1993.

Sobeh, M., Mahmoud, M.F., Abdelfattah, M.A.O., El-Beshbishy, H.A., El-Shazly, A.M.,Wink, M., 2017b. Hepatoprotective and hypoglycemic effects of a tannin richextract from Ximenia americana var. caffra root. Phytomedicine 33, 36–42.

Stintzing, F.C., Herbach, K.M., Mosshammer, M.R., Carle, R., Yi, W., Sellappan, S.,Akoh, C.C., Bunch, R., Felker, P., Color, betalain pattern, and antioxidantproperties of cactus pear (Opuntia spp.) clones. J. Agric. Food Chem. 53, 442–451.

Stintzing, F., Carle, R., 2006. Cactus fruits–more than colour. Fruit Proc. 16, 166–171.van Wyk, B.-E., Wink, M., 2015. Phytomedicines, Herbal Drugs, and Poisons.

University of Chicago Press.Van Wyk, B.-E. and Wink, M., Medicinal Plants of the World, 2017, Briza, Pretoria.White, E., Rao, P., 1953. Constitution of the polysaccharide from tamarind seed. J.

Am. Chem. Soc. 75, 2617–2619.Yáñez, M., Fraiz, N., Cano, E., Orallo, F., 2006. Inhibitory effects of cis-and trans-

resveratrol on noradrenaline and 5-hydroxytryptamine uptake and onmonoamine oxidase activity. Biochem. Biophys. Res. Commun. 344, 688–695.

Youdim, K.A., Dobbie, M.S., Kuhnle, G., Proteggente, A.R., Abbott, N.J., Rice-Evans, C.,2003. Interaction between flavonoids and the blood–brain barrier: in vitrostudies. J. Neurochem. 85, 180–192.

Further Reading

Gawryluk, J.W., Wang, J., Andreazza, A.C., Shao, L., Young, L.T., 2011. Decreasedlevels of glutathione, the major brain antioxidant, in post-mortem prefrontalcortex from patients with psychiatric disorders. Int. J. Neuropsychopharmacol.14, 123–130.

A-MS/MS profiling of secondary metabolites from Opuntia ficus-indica cla-n rats with aluminum chloride induced neurotoxicityOpuntia ficus-indica–